Exhaustive scanning approach to screen all the mitochondrial tRNA genes for mutations and its application to the investigation of 35 independent patients with mitochondrial disorders
Exhaustive scanning approach to screen all the mitochondrial tRNA genes for mutations and its application to the investigation of 35 independent patients with mitochondrial disordersDamien Sternberg1, Claude Danan1, Anne Lombès2, Pascal Laforêt2, Emmanuelle Girodon1, Michel Goossens1 and Serge Amselem1,*
1Service de Biochimie-INSERM U468, Hôpital Henri Mondor, Av. du Maréchal de Lattre de Tassigny, F-94010 Créteil, France and 2INSERM U153, Institut de Myologie, Groupe Hospitalier Pitié-Salpétriêre, Bd de l'Hôpital, 75651 Paris cédex, France
Received July 30, 1997;Revised and Accepted October 9, 1997
To gain a better understanding of the molecular basis of mitochondrial (mt) encephalomyopathies, a highly heterogeneous condition, we developed a denaturing gradient gel electrophoresis-based approach that allows rapid and exhaustive screening for mutations of all 22 mt tRNA-encoding genes and their flanking regions in large cohorts of patients. This method, that detects heteroplasmy (i.e. co-existence of mutant and wild-type mtDNA species in various ratios) directly, was applied to the investigation of 35 independent patients with a disease phenotype compatible with a mitochondrial encephalomyopathy. Twenty-five of the 35 patients investigated displayed a sequence variation in at least one tRNA gene. A total of 46 different sequence variations (41 point mutations, four short insertions and one short deletion), among which 20 are new, were characterized. Forty of them were present in a homoplasmic state, whereas six were heteroplasmic. Twenty-two were located in tRNA genes, among which 10 are new homoplasmic or heteroplasmic sequence variations; 24 were located in flanking regions (12 in mRNA-encoding genes, seven of them leading to missense sequence variations; two in rRNA genes; and 10 in non-coding regions). This study demonstrates (i) the high frequency of homoplasmic tRNA gene sequence variations in our patient sample, and (ii) the existence of several polymorphic sites in tRNA gene regions that may be helpful for defining haplogroups in different populations. It relies on a screening method that can now be applied easily to other population samples.
Human mitochondrial DNA (mtDNA) plays a crucial role in the physiology and pathology of cellular bioenergetics, by participating in the elaboration of the mitochondrial respiratory chain through the synthesis of 13 polypeptides belonging to respiratory complexes I, III, IV and V. Several mtDNA defects affecting cellular function have been described (1 ). The existence of major size rearrangements in mtDNA molecules (2 ) may be responsible for various syndromes, among which chronic progressive external ophthalmoplegia (PEO) is the most frequent. Depletion of mtDNA molecules may cause organ-specific symptoms (3 ). Pathogenic mtDNA point mutations have been found in each type of mtDNA gene (i.e. mRNA, rRNA or tRNA genes). The tRNA gene mutations cause various encephalomyopathy phenotypes including MELAS (myopathy with encephalopathy, lactic acidosis and stroke-like episodes) and MERRF [myoclonic epilepsy with ragged-red fibers (RRF)], associated with the 3243G substitution in the tRNALeuUUR gene and the 8344G substitution in the tRNALys gene, respectively; numerous other familial or sporadic pathological conditions have also been described in association with >30 different point mutations in mt tRNA genes (1 ). Interestingly, most mutations in tRNA genes have been identified in a heteroplasmic state, i.e. co-existence of normal and mutant mtDNAs within the same cell or tissue. The syndromes caused by major size rearrangements or tRNA gene point mutations are usually associated with muscular histopathological abnormalities, such as RRF or mosaic deficiency of cytochrome oxidase activity (i.e. COX-negative fibers) (4 ). However, in several patients presenting with such features, the search for a major rearrangement and already described point mutations in tRNA genes remains negative (5 ), making it necessary to explore the complete sequence of all 22 tRNA genes. With this aim, we designed optimal experimental conditions to detect mt tRNA mutations in a simple, rapid, sensitive and reliable way, using denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified fragments. Thirty-five independent patients presenting with encephalopathy or myopathy associated with ragged-red or COX-negative fibers were investigated following this procedure.
Muscular mtDNA was analyzed in all the 35 patients whose clinical and biological features are summarized in Table 1 . A DGGE-based screening system (6 ) using PCR-amplified fragments containing a GC-rich tail (7 ,8 ) was designed by means of computational simulation using the algorithms provided by Lerman et al. (9 ). Complete scrutiny of mt tRNA genes was warranted by the location of these genes in the lowest melting domain at one end of the amplification fragments (8 ,10 ), and by use of electrophoretic conditions allowing optimal discrimination between homoduplexes and heteroduplexes, as predicted by computational analysis (9 ). The nucleotide sequences of the primers and the electrophoretic conditions are provided in Table 2 .
In all but one PCR fragment, the efficiency of mutation detection was illustrated by the detection of heteroduplexes that were generated either spontaneously in heteroplasmic patients, or after mixing different PCR samples in homoplasmic patients (see Materials and Methods). Several variants were identified in each PCR fragment, except in fragment 5 (tRNATrp region) where no sequence variation was detected in the 35 patients investigated (Table 3 ). The number of sequence variants and their relative frequency in our patient population differed widely from region to region. Table 0 summarizes which changes were found in which patient.
RRF, ragged-red fibers; s, sporadic case; oc, other case(s) within the family compatible with maternal inheritance; PEO, progressive external ophthalmoplegia; DIDMOAD, diabetes insipidus, diabetes mellitus, optic atrophy and deafness; MNGIE, myopathy, neuropathy, gastrointestinal dysmotility and encephalopathy; + , present; -, absent. aPatient 9 was suspected with mitochondrial disease when giant mitochondria were detected in circulating lymphocytes originating from a splenic lymphoma.
Specific conditions of amplification and DGGE analysis of the 15 fragments encompassing tRNA genes
ID of amplified fragment
tRNA genes explored
Specific conditions for amplification
Specific conditions for DGGE
Primers (length of additional GC tail at 5' end)
T° of annealing (no. of cycles)
Denaturing gradient in the gel (top-bottom)
Duration of electrophoresis (h)
1
Phe
F529-548 (60) R700-719
60°C (40)
10-60%
4
2
Val
F1487-1506 (35) R1695-1714
55°C (40)
10-60%
4
3
LeuUUR
F3163-3182 R3401-3420 (35)
55°C (40)
10-60%
4
4
Ile, Gln, Met
F4242-4261 R4538-4560 (30)
55°C (40)
10-60%a 25-75%a
4a 8a
5
Trp
F5470-5489 (30) R5583-5602
61°C (35)
10-60%
3.5
6
Ala, Asn
F5541-5561 R5743-5762 (40)
64°C (45)
10-60%
4
7
Cys, Tyr
F5741-5760 R5898-5917 (40)
61°C (35)
10-60%
2.5
8
SerUCN
F7409-7428 R7529-7553 (45)
55°C (40)
10-60%
4
9
Asp
F7493-7512 R7619-7639 (20)
55°C (40)
10-60%
2.25
10
Lys
F8128-8147 (7) R8538-8557
55°C (40)
10-60%
10
11
Gly
F9912-9931 (40) R10 143-10 162
55°C (40)
10-60%
4
12
Arg
F10 298-10 317 (40) R10 528-10 547
55°C (40)
10-60%
4
13
His, SerAGY, LeuCUN
F12 115-12 134 (40) R12 347-12 367 (10)
61°C (35)
10-60%
4
14
Glu
F14 636-14 655 (40) R14 842-14 861
55°C (40)
10-60%
4
15
Thr, Pro
F15 828-15 848 (39) R16 079-16 098
55°C (40)
10-60%
4
F, forward primer; R, reverse primer. aTwo electrophoreses are necessary to explore the tRNA genes included in fragment 4: one short migration in mild denaturing conditions to explore the first melting domain containing the tRNAIle gene; one longer migration in stronger denaturing conditions to explore the second melting domain containing tRNAGln and tRNAMet genes.
Forty-six different sequence variations (41 point mutations, four short insertions and one short deletion), among which 20 are new, were characterized. Forty of them were present in a homoplasmic state, whereas six of them were in a heteroplasmic state (gain of three Cs at 568-573, gain of seven Cs at 568-573, 3243G, 4450A, 5692C, 8344G). Twenty-two are located in tRNA genes and 24 in flanking regions (10 in non-coding regions, two in rRNA genes and 12 in mRNA-encoding genes, among which seven are missense sequence variations and five synonymous substitutions) (Table 3 ).
Six different heteroplasmic sequence variations (as revealed by multiple-band patterns), among which two are new, were identified in 13 different patients. Four of them are located in tRNA genes, whereas the two remaining variations consist of short insertions of different sizes in a polypyrimidine tract at position 568-573 in the D-loop region, near the tRNAPhe gene (Table 3 ).
Seven patients (patients 1-7) with various clinical phenotypes (Table 1 ) presented with heteroplasmic profiles in the tRNALeuUUR region (fragment 3, Fig. 1 A). In all cases, the sequence variation responsible for heteroplasmy was the 3243G mutation (MELAS mutation), as characterized by enzymatic digestion. However, two of these patients presented with a different DGGE migration pattern; sequencing revealed an additional sequence variation in a homoplasmic state in the ND1 gene: the synonymous 3336C substitution (patient 4) and the 3316A variation leading to a A4T missense mutation (patient 6, Fig. 1 A).
One patient presenting with MERRF syndrome (patient 8) displayed a heteroplasmic DGGE profile in the tRNALys region (fragment 10, Fig. 1 B). As expected, PCR restriction analysis revealed the 8344G point mutation usually associated with this disease (11 ).
Summary of mtDNA sequence variations characterized in this study
Fragment (number of patients analyzed)
Nucleotide sequence
mtDNA region(s) affected
Number of patients
1 (27)
Reference sequence (H)
23
Gain of one C at 568-573 (H)
nc
1
Gain of three Cs at 568-573 (h)
nc
1
Gain of seven Cs at 568-573 (h)
nc
2
2 (27)
Reference sequence
25
1536G (H)
12S rRNA
1
1664A (H)
tRNAVal
1
3 (35)
Reference sequence (H)
22
3243G (h)
tRNALeuUUR
5
3243G (h)/3316A (H)
tRNALeuUUR/ND1 (A4T)
1
3243G (h)/3336C (H)
tRNALeuUUR/ND1 (syn)
1
3398C (H)
ND1 (M31T)
1
3197C (H)
16S rRNA
5
4 (27)
Reference sequence (H)
26
4450A (h)
tRNAMet
1
5 (27)
Reference sequence (H)
27
6 (27)
Reference sequence (H)
23
5583T (H)
nc
1
5633T (H)
tRNAAla
1
5656G (H)
nc
1
5692C (h)
tRNAAsn
1
7 (27)
Reference sequence (H)
25
Gain of one C at 5895-5899 (H)
nc
1
5814C (H)
tRNACys
1
8 (27)
Reference sequence (H)
25
7476T (H)
tRNASerUCN
1
7430G (H)
COX1 (syn)
1
9 (27)
Reference sequence (H)
26
7581C (H)
tRNAAsp
1
10 (28)
Reference sequence (H)
25
9 bp deletion at 8272-8280 (H)
nc
1
8344G (h)
tRNALys
1
8348G (H)
tRNALys
1
11 (27)
Reference sequence (H)
22
10 014A (H)
tRNAGly
1
10 034C (H)
tRNAGly
2
10 042G (H)
tRNAGly
1
10 103G (H)
ND3 (syn)
1
12 (27)
Reference sequence (H)
16
10 398G (H)/10 400T (H)
ND3 (T114A)
1
10 398G (H)
ND3 (T114A)
5
10 463C (H)
tRNAArg
3
10 506G (H)
ND4L (T13A)
1
10 398G (H)/10 410C (H)/10 457C (H)
ND3 (T114A)/tRNAArg
1
Table 3.Continued
Fragment (number of patients analyzed)
Nucleotide sequence
mtDNA region(s) affected
Number of patients
13 (27)
Reference sequence (H)
21
12 308G (H)
tRNALeuCUN
5
12 234G (H)
tRNASerAGY
1
14 (27)
Reference sequence (H)
10
14 766C (H)
CytB (I7T)
11
14 783C (H)
CytB (syn)
1
14 798C (H)
CytB (F18L)
3
14 793G (H)
CytB (H16R)
2
15 (27)
Reference sequence (H)
14
15 904T (H)
tRNAThr
1
15 928A (H)
tRNAThr
3
16 069T (H)
nc
3
15 913T (H)/16 069T (H)
tRNAThr/nc
1
15 924G (H)
tRNAThr
2
15 954C (H)
nc
1
16 051G (H)
nc
2
h: sequence variation found in a heteroplasmic state; H: sequence variation found in a homoplasmic state; nc: non-coding region. Previously undescribed mutations are shown in bold characters. All nucleotide substitutions are reported as L-strand changes.
Previously undescribed mutations are shown in bold. Sequence variations reported for the second time are shown in normal characters. Sequence variations previously described in healthy subject populations as frequent polymorphisms (frequency of at least 1%) are shown in italics. syn: synonymous sequence variation.
Location in tRNA and phylogenetic conservation of mutated nucleotides for 10 rare homoplasmic tRNA sequence variations
Sequence variation
tRNA
Localization of the substitution in tRNA
Phylogenetic conservation of substituted nucleotide
Phylogenetic conservation of
1664A
Val
acceptor arm
low
nr
5633T
Ala
junction of the stems of D arm and anticodon arm
middle
nr
5814Ca
Cys
stem of D arm
middle
high
7581C
Asp
acceptor arm
low
nr
8348G
Lys
T[psi]C loop
low
nr
10014A
Gly
stem of anticodon arm
middle
high
10042G
Gly
T[psi]C loop
low
nr
10457C
Arg
stem of T[psi]C arm
middle
high
12234G
Ser AGY
stem of anticodon arm
middle
nr
15913T
Thr
stem of anticodon arm
low
high
nr: non-relevant (no Watson-Crick pairing at this site).
aPreviously reported as pathogenic (see ref. 14).
Another patient (patient 9), presenting with a splenic lymphoma, displayed a heteroplasmic profile in the tRNAsIle,Gln,Met region (fragment 6), due to the 4450A sequence variation in the tRNAMet gene. The mutated species, which was identified as the predominant species in circulating leukocytes (Fig. 1 C), was also found in muscle as the minor species (12 ).
A heteroplasmy in the tRNAsAla,Asn gene region (fragment 6) was discovered in a 55-year-old patient (patient 10) with PEO, ataxia, deafness, peripheral neuropathy and hypertrophic cardiomyopathy. Sequencing revealed a point mutation in the tRNAAsn gene (5692C) that was reported previously in a patient with a similar clinical pattern (13 ). Mutated and wild-type mtDNA species were equally represented, as reflected by the similar intensity of each homoduplex band (Fig. 1 D).
Three patients carried a heteroplasmic sequence variation in the tRNAPhe gene region (fragment 1). This heteroplasmy is actually due to the insertion of several cytosines (Cs) in the polyC tract at position 568-573, which is a part of the non-coding D-loop region: we found three more Cs in patient 17, and seven more Cs in patients 25 and 29.
Forty different homoplasmic sequence variations (as revealed by single band patterns), among which 18 are new, were characterized: 18 were located in the tRNA genes and 22 in the surrounding regions (12 in structural genes, two in rRNA genes and eight in non-coding regions) (Table 3 ).
Homoplasmic variations in tRNA-encoding genes. Eighteen different homoplasmic variations were found in the tRNA genes. Nine of them are new: 1664A in tRNAVal and 10014A in tRNAGly (patient 18); 5633T in tRNAAla (patient 22); 7581C in tRNAAsp (patient 16); 8348G in tRNALys (patient 28); 10042G in tRNAGly (patient 21); 12234G in tRNASerAGY (patient 33); 10457C in tRNAArg and 15913T in tRNAThr (patient 9). The previously described 5814C substitution in the tRNACys gene (14 ) was found in one patient (patient 27) in a homoplasmic state (Fig. 1 E). In Table 5 the location of these 10 mutated nucleotides in tRNA and their phylogenetic conservation are presented. The other eight variations are frequent polymorphisms that were described previously in control subjects at a frequency of at least 1%: 7476T in tRNASerUCN (15 ), 10034C in tRNAGly (16 ), 12308G in tRNALeuCUN (17 ), 10410C in tRNAArg (18 ), 15904T (15 ) and 15924G (19 ) in tRNAThr, 10463C in tRNAArg and 15928A in tRNAThr, the two latter being usually associated (15 ).
Homoplasmic variations in the tRNA gene flanking regions. In the course of the identification of mt tRNA gene mutations, we detected sequence variations in the flanking regions. Twenty-two different sequence variations, among which 10 are new, were identified: 19 substitutions, two short insertions and one short deletion. Twelve occurred in mRNA-encoding regions, one in the 12S rRNA gene, one in the 16S rRNA gene and eight in the non-coding regions (Table 3 ).
Seven of the 12 sequence variations located in protein subunit genes consist of missense variations. Two of them are new: 3398C (M31T) in ND1, which is associated with the heteroplasmic 4450A mutation in tRNAMet (patient 9), and 14793G (H16R) in cytochrome b (patients 25 and 26). The other missense variations found in our patient sample were: the previously reported 3316A leading to a A4T amino acid substitution in ND1 (20 ); 10398G, a frequent polymorphism leading to a T114A substitution in ND3 (21 ); 10506G leading to a T13A substitution in ND4 (22 ); and 14766C and 14798C, two frequent polymorphisms leading to the I7T and F18L substitutions in cytochrome b (16 ,23 ). The five synonymous substitutions were 3336C in ND1, 7430G in COX1, 10103G and 10400T in ND3 and 14783C in cytochrome b.
The homoplasmic variations found in the 12S and 16S rRNA genes were 1536G (patient 31) and 3197C (patients 21, 25, 26, 27, 34), respectively; it is likely that the 16S rRNA gene variation is a frequent Caucasian polymorphism, as it was found with the same frequency in our patient and control populations (data not shown). Homoplasmic variations found in the non-coding regions were gain of one C at 568-573 (patient 21); 5583T, in the short non-coding stretch between tRNATrp and tRNAAla genes (patient 25); 5656G, changing the non-coding base pair between tRNAAla and tRNAAsn genes (patient 27); gain of one C at 5895-5899, in the short non-coding region between the tRNAAsn and COX1 genes (patient 28); the frequently described 9- bp deletion at 8272-8280 (24 ,25 ) (patient 21); 15954C, changing the non-coding base pair between tRNAThr and tRNAPro genes (patient 16); 16051G (patients 27 and 31) and 16069T (patients 9, 19, 22 and 23) in the D-loop region.
Looking for point mutations in mtDNA is a hard task. First, such mutations may occur at any position, thereby underlining the need to develop exhaustive screening methods; second, after detecting and identifying new sequence variations, classifying them as polymorphisms or pathogenic mutations may be difficult: one needs especially to know if they are present in a homoplasmic or heteroplasmic state. Among the different screening techniques that have been proposed to study mtDNA (26 -32 ), a DGGE-based approach using a GC-clamp and heteroduplex detection is a powerful way to overcome these two difficulties. Indeed, it relies on a screening method that is exhaustive in optimal experimental conditions (10 ,33 ); in addition, it allows direct detection of a minor heteroplasmy that can escape standard molecular analyses based on direct sequencing or on the cloning of PCR-amplified fragments. The efficiency of such a screening procedure is attested to by the very large number of nucleotide variations identified in this study (Table 3 ).
A heteroplasmic DGGE pattern was found in 13 out of the 35 patients investigated. In 10 of these patients, the heteroplasmy was due to a pathogenic sequence variation in a tRNA-encoding gene. The 3243G mutation in the tRNALeuUUR gene, usually associated with MELAS, PEO or diabetes and deafness, was found in six patients presenting with these phenotypes, and in one patient (patient 3) presenting with an atypical encephalomyopathy. The 8344G mutation in the tRNALys gene, usually associated with MERRF, was found in one MERRF patient. The 4450A mutation in the tRNAMet gene and the previously described 5692C mutation in the tRNAAsn gene were found in one patient each. At first glance, these two mutations are very likely to be deleterious, as they change evolutionarily conserved nucleotides that are involved in the structure of the T arm of tRNAMet and the anticodon arm of tRNAAsn, respectively (12 ,13 ). Although the relevance of the 4450A mutation to patient 9's lymphoma remains unclear, its pathogenic character was indeed confirmed by the study of respiratory phenotype of transmitochondrial cybrids (12 ).
However, heteroplasmy is not an absolute hallmark of pathogenicity. Indeed, in three patients and controls, we found heteroplasmic patterns in the tRNAPhe gene region due to insertions of several Cs in the polyC tract at position 568-573. Analogous insertions of 2-6 Cs at the same site were described previously as non-pathogenic polymorphisms associated with heteroplasmy in a Caucasian haplogroup (34 ).
Heteroplasmy is a transitional state; it results from the occurrence of a mtDNA point mutation, and may lead to its fixation in a homoplasmic state in one cell, one tissue or one individual, through segregation of the mutated mtDNA species, either at random or under nuclear control (35 ). The more deleterious the mutation is, the higher is the constraint exerted against the fixation of the mutated species in a homoplasmic state (36 ). A homoplasmic state is, therefore, an argument against a strong pathogenic character of a mitochondrial sequence variation. However, this does not rule out a mildly deleterious consequence with a possible additive or synergistic effect in combination with other homoplasmic (37 ) or heteroplasmic point mutations, or major size rearrangements (38 ). This might be the case for several rare homoplasmic sequence variations identified in our patient population and located in the tRNA genes and their flanking regions. Indeed, four of these sequence variations were found in association with a heteroplasmic pathogenic mutation, raising the question of their contribution to the disease phenotype. The A4T substitution in the ND1 gene, which has been reported in patients with non-insulin-dependent diabetes mellitus (20 ), was found in association with the 3243G mutation in patient 6 who presented with PEO and strokes. The M31T missense variation in the ND1 gene and two other nucleotide substitutions (10457C and 15913T in tRNAArg and tRNAThr genes, respectively) were found together with the 4450A mutation in patient 9 presenting with a splenic lymphoma. In addition, two homoplasmic missense variations, that so far have been identified only in pathological situations, were found in three patients: the new H16R substitution (14793G) in the cytochrome b of patients 25 and 26 presenting with two different abnormal phenotypes (Table 1 ), and the T13A missense variation (10506G) in the ND4L gene, which was identified in patient 20 presenting with PEO, and previously reported in one LHON patient (22 ).
The 5814C substitution in the tRNACys gene, found in patient 27, a 60-year-old woman presenting with mild late-onset myopathy, is likely to be deleterious. According to the DGGE pattern, the muscular DNA of this patient carried the mutation in a homoplasmic state. Strikingly, the same mutation was described recently in a heteroplasmic state in the muscle of a 5-year-old child presenting with a MELAS-like syndrome (14 ). Several hypotheses may explain the association of two very different phenotypes with this mutation, such as a different distribution of wild-type and mutated species in tissues of the two patients and an organ-specific pathogenicity, or the existence of various mtDNA or nuclear backgrounds.
For several other rare homoplasmic sequence variations found in the tRNA genes of our patient sample, a pathogenic contribution to the disease phenotype could not be excluded, although evaluation of phylogenetic conservation of the mutated nucleotides produced no argument for their pathogenic role (Table 5 ).
In conclusion, this study demonstrates the high frequency of homoplasmic sequence variations in human mt tRNA genes and underlines the molecular heterogeneity of mitochondrial neuromuscular disorders when no major size rearrangement of mtDNA is detected. The system used in the present study to screen for mt tRNA sequence variations allows a rapid detection of heteroplasmic and homoplasmic mutations in large groups of patients. It can now be applied easily to other patient and control populations. It may also be helpful for defining haplogroups in different populations, given its efficiency in detecting polymorphic sites.
Thirty-five independent patients were referred to us to investigate a possible mitochondrial disease that was suspected on the basis of clinical and biological features and familial inquiry (Table 1 ). In most cases (31/35), histological and histochemical examination of a skeletal muscle biopsy revealed RRF or COX-negative fibers. No major mtDNA rearrangement was detected by Southern blotting in all 35 patients.
In all patients, mtDNA was extracted from muscular biopsy specimens, as described (39 ). In one case (patient 9), mtDNA was also obtained from white blood cells following standard techniques.
The melting behavior of double-stranded DNA fragments encompassing mt tRNA genes was analyzed by means of the MELT87 program (9 ). This information was used to select PCR primers to amplify fragments suitable for DGGE. As the sequence of interest has to be located within the first melting domains of the fragment, a GC-rich tail of variable length was added at the 5' end of one of the two primers (8 ). Optimal experimental conditions (i.e. denaturing gradient, voltage, time length) to discriminate heteroduplexes from homoduplexes were determined for each selected fragment by means of the SQHTX program (9 ). Fifteen amplification fragments, numbered 1-15, were selected to allow exhaustive DGGE screening of the 22 tRNA genes (Table 2 ). The corresponding melting curves are available upon request.
PCR was used to obtain a high copy number of selected mt DNA regions. It was performed in a 100 µl reaction mixture containing 100 ng of total DNA as template, 0.2 pmol/µl of each primer, dNTP at a concentration of 200 µM for each and 5 U of Taq polymerase (ATGC Biotechnologies, Noisy-le-Grand, France). The amplification conditions specific to each fragment are summarized in Table 4 .
DGGEs were run in a Tris-EDTA-acetate buffer (TEA: Tris 40 mM; Na acetate 20 mM; EDTA 1 mM; H2O qsp), at 60°C, using a 6% polyacrylamide gel containing a gradient of denaturing agents (100% denaturant = 7 M urea and 40% formamide). The gels were stained with ethidium bromide and examined under UV light.
The double-stranded DNA products generated for DGGE analysis were subjected to direct sequencing using an ABI 373A automated DNA sequencer (Perkin-Elmer Corp., Applied Biosystems Division). We used the updated Cambridge human mtDNA light strand sequence as the reference sequence (40 ,41 ).
Given the frequent occurrence of mutations in tRNALeuUUR and tRNALys genes, the two PCR fragments encompassing these genes (regions 3 and 10, respectively) were first explored in all patients. Samples that displayed heteroplasmy in region 3 were screened for the presence of the 3243G mutation in the tRNALeuUUR gene by restriction analysis using HaeIII. Patients that showed heteroplasmy in region 10 were tested for the most frequent mutation in the tRNALys gene (8344G) by enzymatic digestion of an amplified fragment generated with primer F8128-8147 (Table 2 ) and a reverse primer (5'>GGG GCA TTT CAC TGT AAA GAG GTG TTA G<3'), the 3' sequence of which was modified to create an AluI restriction site in the presence of the 8344G mutation. When no heteroplasmic mutation was detected in fragments 3 and 10, patients were submitted to exhaustive DGGE analysis in all regions.
As the mutated species was predominant in all heteroplasmic patterns, characterization of the underlying mutation could be achieved by direct sequencing. In each region, samples with different homoplasmic DGGE patterns (i.e. different migration levels) were easily distinguished; one sample corresponding to each migration level was sequenced. PCR samples displaying identical DGGE patterns were mixed together in groups of four samples, and analyzed subsequently by DGGE after a cycle of melting and reassociating: the presence of heteroduplexes pointed to the existence of a sample with an original sequence, that was identified further by direct sequencing.
This study was supported by grants from the Institut National de la Santé et de la Recherche Médicale (INSERM), the Association Française contre les Myopathies, and the Groupe de Recherche et d'Etude sur le Génome (GREG).
1Suomalainen, A. (1997) Mitochondrial DNA and disease. Ann. Med., 29, 235-246.MEDLINE Abstract
2Holt, I.J., Harding, A.E. and Morgan-Hughes, J.A. (1988) Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies. Nature, 331, 717-719.MEDLINE Abstract
3Moraes, C.T., Shanske, S., Tritschler, H.J., Aprille, J.R., Andreetta, F., Bonilla, E., Schon, E.A. and DiMauro, S. (1991) MtDNA depletion with variable tissue expression: a novel genetic abnormality in mitochondrial diseases. Am. J. Hum. Genet., 48, 492-501.MEDLINE Abstract
4Shoubridge, E.A. (1994) Mitochondrial DNA diseases: histological and cellular studies. J. Bioenerg. Biomembr., 26, 301-310.MEDLINE Abstract
5Laforêt, P., Lombès, A., Eymard, B., Danan, C., Chavallay, M., Rouche, A., Frachon, P. and Fardeau, M. (1995) Chronic progressive external ophtalmoplegia with ragged-red fibers: clinical, morphological and genetic investigations in 43 patients. Neuromusc. Disord., 5, 399-413.MEDLINE Abstract
6Fodde, R. and Losekoot, M. (1994) Mutation detection by denaturing gradient gel electrophoresis (DGGE). Hum. Mutat., 3, 83-94.MEDLINE Abstract
7Myers, R.M., Fischer, S.G., Maniatis, T. and Lerman, L.S. (1985) Modification of the melting properties of duplex DNA by attachment of a GC-rich DNA sequence as determined by denaturing gradient gel electrophoresis. Nucleic Acids Res., 13, 3111-3129.MEDLINE Abstract
8Sheffield, V.C., Cox, D.R., Lerman, L.S. and Myers, R.M. (1989) Attachment of a 40-base G+C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc. Natl Acad. Sci. USA, 86, 232-236.MEDLINE Abstract
9Lerman, L.S. and Silverstein, K. (1987) Computational simulation of DNA melting and its application to denaturing gradient gel electrophoresis. Methods Enzymol., 155, 482-501.MEDLINE Abstract
10Abrams, E., Murdaugh, S.E. and Lerman, L.S. (1990) Comprehensive detection of single base changes in human genomic DNA using denaturing gradient gel electrophoresis and a GC clamp. Genomics, 7, 463-475.MEDLINE Abstract
11Shoffner, J.M., Lott, M.T., Lezza, A.M.S., Seibel, P., Ballinger, S.W. and Wallace, D.C. (1990) Myoclonic epilepsy and ragged-red fiber disease (MERRF) is associated with a mitochondrial DNA tRNALys mutation. Cell, 61, 931-937.MEDLINE Abstract
12Lombès, A., Bories, D., Girodon, E., Frachon, P., Ngo, M.M., Breton-Gorius, J., Tulliez, M. and Goossens, M. (1997) The first pathogenic mitochondrial methionine tRNA point mutation is discovered in splenic lymphoma. Hum. Mutat., in press.
13Seibel, P., Lauber, J., Klopstock, T., Marsac, C., Kadenbach, B. and Reichmann, H. (1994) Chronic progressive external ophtalmoplegia is associated with a novel mutation in the mitochondrial tRNAAsn gene. Biochem. Biophys. Res. Commun., 204, 482-489.MEDLINE Abstract
14Manfredi, G., Schon, E.A., Bonilla, E., Moraes, C.T., Shanske, S. and DiMauro, S. (1996) Identification of a mutation in the mitochondrial tRNACys gene associated with mitochondrial encephalopathy. Hum. Mutat., 7, 158-163.MEDLINE Abstract
15Houshmand, M., Larrson, N., Holme, E., Oldfors, A., Tulinius, M.H. and Andersen, O. (1994) Automatic sequencing of mitochondrial tRNA genes in patients with mitochondrial encephalomyopathy. Biochim. Biophys. Acta, 1226, 49-55.MEDLINE Abstract
16Marzuki, S., Noer, A.S., Lertrit, P., Thyagarajan, D., Kapsa, R., Utthanaphol, P. and Byrne, E. (1991) Normal variants of human mitochondrial DNA and translation products: the building of a reference database. Hum. Genet., 88, 139-145.MEDLINE Abstract
17Van den Ouweland, J.M.W., Bruining, G.J., Lindhout, D., Wit, J., Veldhuyzen, B.F.E. and Maassen, J.A. (1992) Mutations in mitochondrial tRNA genes: non-linkage with syndromes of Wolfram and chronic progressive external ophtalmoplegia. Nucleic Acids Res., 20, 679-682.
18Hattori, Y., Goto, Y., Sakuta, R., Nonaka, I., Mizuno, Y. and Horai, S. (1994) Point mutations in mitochondrial tRNA genes: sequence analysis of chronic progressive external ophtalmoplegia (CPEO). J. Neurol. Sci., 125, 50-55.MEDLINE Abstract
19Brown, M.D., Torroni, A., Schoffner, J.M. and Wallace, D.C. (1992) Mitochondrial tRNAThr mutations and lethal infantile mitochondrial myopathy. Am. J. Hum. Genet., 51, 446-447.MEDLINE Abstract
20Nakagawa, Y., Ikegami, H., Yamato, E., Takekawa, K., Fujisawa, T., Hamada, Y., Ueda, H., Uchigata, Y., Tetsuro, M., Kumahara, Y. and Ogihara, T. (1995) A new mitochondrial DNA mutation associated with non-insulin-dependent diabetes mellitus. Biochem. Biophys. Res. Commun., 209, 664-668.MEDLINE Abstract
21Stoneking, M., Hedgecock, D., Higuchi, R.G., Vigilant, L. and Erlich, H.A. (1991) Population variation of human mtDNA control region sequences detected by enzymatic amplification and sequence-specific oligonucleotide probes. Am. J. Hum. Genet., 48, 370-382.MEDLINE Abstract
22Howell, N., Bindoff, L.A., McCullough, D.A., Kubacka, I., Poulton, J., Mackey, D., Taylor, L. and Turnbull, D.M. (1991) Leber hereditary optic neuropathy: identification of the same mitochondrial ND1 mutation in six pedigrees. Am. J. Hum. Genet., 49, 939-950.MEDLINE Abstract
23Mackey, D. and Howell, N. (1992) A variant of Leber hereditary optic neuropathy characterized by recovery of vision and by an unusual mitochondrial genetic etiology. Am. J. Hum. Genet., 51, 1218-1228.MEDLINE Abstract
24Wrischnik, L.A., Higuchi, R.G., Stoneking, M., Erlich, H.A., Arnheim, N. and Wilson, A.C. (1987) Length mutations in human mitochondrial DNA: direct sequencing of enzymatically amplified DNA. Nucleic Acids Res., 15, 530-541.
25Soodyall, H., Vigilant, L., Hill, A.V., Stoneking, M. and Jenkins, T. (1996) MtDNA control-region sequence variation suggests multiple independent origins of an `asian-specific' 9-bp deletion in sub-saharan Africans. Am. J. Hum. Genet., 58, 595-608.MEDLINE Abstract
27Yoon, K.L., Modica-Napolitano, J.S., Ernst, S.G. and Aprille, J.R. (1991) Denaturing gradient gel method for mapping single base changes in human mitochondrial DNA. Anal. Biochem., 196, 427-432.MEDLINE Abstract
28Suomalainen, A., Ciafaloni, E., Koga, Y., Peltonen, L., DiMauro, S. and Schon, E.A. (1992) Use of single strand conformation polymorphism analysis to detect point mutations in human mitochondrial DNA. J. Neurol. Sci., 111, 222-226.MEDLINE Abstract
29Yoon, K.L., Brown, M.D. and Wallace, D.C. (1994) Single-strand conformation polymorphism analysis for the detection of point mutations in the mitochondrial DNA. Anal. Biochem., 224, 608-611.
30Thomas, A.W., Morgan, R., Sweeney, M., Rees, A. and Alcolado, J. (1994) The detection of mitochondrial DNA mutations using single stranded conformation polymorphism (SSCP) analysis and heteroduplex analysis. Hum. Genet., 94, 621-623.MEDLINE Abstract
31Gross, A.W., Aprille, J.R. and Ernst, S.G. (1994) Identification of human mitochondrial DNA fragments corresponding to the genes for ATPase, cytochrome c oxidase, and nine tRNAs in a denaturing gradient gel electrophoresis system. Anal. Biochem., 222, 507-510.MEDLINE Abstract
32Hanekamp, J.S., Thilly, W.G. and Ahmad Chaudhry, M. (1996) Screening for human mitochondrial DNA polymorphisms with denaturing gradient gel electrophoresis. Hum. Genet., 98, 243-245.MEDLINE Abstract
33Dianzani, I., Camaschella, C., Ponzone, A. and Cotton, R.G.H. (1993) Dilemmas and progress in mutation detection. Trends Genet., 9, 403-405.MEDLINE Abstract
34Torroni, A., Lott, M.T., Cabell, M.F., Chen, Y., Lavergne, L. and Wallace, D.C. (1994) mtDNA and the origin of Caucasians: identification of ancient Caucasian-specific haplogroups, one of which is prone to a recurrent somatic duplication in the D-loop region. Am. J. Hum. Genet., 55, 760-776.MEDLINE Abstract
35Dunbar, D.R., Moonie, P.A., Jacobs, H.T. and Holt, I.J. (1995) Different cellular backgrounds confer a marked advantage to either mutant or wild-type mitochondrial genomes. Proc. Natl Acad. Sci. USA, 92, 6562-6566.MEDLINE Abstract
36Wallace, D.C. (1995) 1994 William Allan award address: mitochondrial DNA variation in human evolution, degenerative disease, and aging. Am. J. Hum. Genet., 57, 201-223.MEDLINE Abstract
37Johns, D.R. and Berman, J. (1991) Alternative simultaneous complex I mitochondrial DNA mutations in Leber's hereditary optic neuropathy. Biochem. Biophys. Res. Commun., 174, 1324-1330.MEDLINE Abstract
38Reynier, P., Figarella-Branger, D., Serrtrice, G., Charvet, B. and Malthièry, Y. (1994) Association of deletion and homoplasmic point mutation of the mitochondrial DNA in an ocular myopathy. Biochem. Biophys. Res. Commun., 202, 1606-1611.MEDLINE Abstract
39Zeviani, M., Moraes, C.T., DiMauro, S., Nakase, H., Bonilla, E., Schon, E.A. and Rowland, L.P. (1988) Deletions of mitochondrial DNA in Kearns-Sayre syndrome. Neurology, 38, 1339-1346.MEDLINE Abstract
40Anderson, S., Bankier, A.T., Barrell, B.G., De Bruijn, M.H.L., Coulson, A.R., Drouin, J., Eperon, I.C., Nierlich, D.P., Roe, B.A., Sanger, F., Schreier, P.H., Smith, A.J.H., Staden, R. and Young, I.G. (1981) Sequence and organization of the human mitochondrial genome. Nature, 290, 457-465.MEDLINE Abstract
41Howell, N., McCullough, D.A., Kubacka, I., Halvorson, S. and Mackey, D. (1992) The sequence of human mtDNA: the question of errors versus polymorphisms. Am. J. Hum. Genet., 50, 1333-1337.MEDLINE Abstract
*To whom correspondence should be addressed. Tel: +33 1 49812873; Fax: +33 1 49812842; Email: amselem@im3.inserm.fr
K. S. Lim, R. K. Naviaux, and R. H. Haas Quantitative Mitochondrial DNA Mutation Analysis by Denaturing HPLC
Clin. Chem.,
June 1, 2007;
53(6):
1046 - 1052.
[Abstract][Full Text][PDF]
K. Aure, G. Fayet, J. P. Leroy, E. Lacene, N. B. Romero, and A. Lombes Apoptosis in mitochondrial myopathies is linked to mitochondrial proliferation
Brain,
May 1, 2006;
129(5):
1249 - 1259.
[Abstract][Full Text][PDF]
C. T. Moraes, D. P. Atencio, J. Oca-Cossio, and F. Diaz Techniques and Pitfalls in the Detection of Pathogenic Mitochondrial DNA Mutations
J. Mol. Diagn.,
November 1, 2003;
5(4):
197 - 208.
[Abstract][Full Text][PDF]
L.-J. C. Wong, M.-H. Liang, H. Kwon, J. Park, R.-K. Bai, and D.-J. Tan Comprehensive Scanning of the Entire Mitochondrial Genome for Mutations
Clin. Chem.,
November 1, 2002;
48(11):
1901 - 1912.
[Abstract][Full Text][PDF]
M. Jaksch, S. Kleinle, C. Scharfe, T. Klopstock, D. Pongratz, J. Muller-Hocker, K.-D Gerbitz, S. Liechti-Gallati, H. Lochmuller, and R. Horvath Frequency of mitochondrial transfer RNA mutations and deletions in 225 patients presenting with respiratory chain deficiencies
J. Med. Genet.,
October 1, 2001;
38(10):
665 - 673.
[Abstract][Full Text][PDF]
C. Karadimas, K. Tanji, M. Geremek, P. Chronopoulou, T. Vu, S. Krishna, C. M. Sue, S. Shanske, E. Bonilla, S. DiMauro, et al. A5814G Mutation in Mitochondrial DNA Can Cause Mitochondrial Myopathy and Cardiomyopathy
J Child Neurol,
July 1, 2001;
16(7):
531 - 533.
[Abstract][PDF]
D. Sternberg, E. Chatzoglou, P. Laforet, G. Fayet, C. Jardel, P. Blondy, M. Fardeau, S. Amselem, B. Eymard, and A. Lombes Mitochondrial DNA transfer RNA gene sequence variations in patients with mitochondrial disorders
Brain,
May 1, 2001;
124(5):
984 - 994.
[Abstract][Full Text][PDF]
M. Bataillard, E. Chatzoglou, L. Rumbach, D. Sternberg, A. Tournade, P. Laforet, C. Jardel, T. Maisonobe, and A. Lombes Atypical MELAS syndrome associated with a new mitochondrial tRNA glutamine point mutation
Neurology,
February 13, 2001;
56(3):
405 - 407.
[Abstract][Full Text][PDF]
CiteULike 
Connotea 
Del.icio.us What's this?
